DESCRIPTION (Applicant's Description): Apoptosis or programmed cell death eliminates redundant or damaged cells in multicellular organisms. Abnormal apoptosis contributes to the pathogenesis of many human diseases including cancer. Bcl-2 and related proteins function as either suppressors or promoters of apoptosis. Aberrant expression of them leads to dysregulation of apoptosis and to the onset of apoptotic diseases. Bcl-2 is anchored on the membrane of the mitochondrion, the endoplasmic reticulum (ER) or the nucleus via its C-terminal transmembrane sequence. Bcl-2 maintains the normal function of the membranes in many apoptotic events, protecting the organelles and hence the cells. The structure of the cytosolic domain of Bcl-XL, a Bcl-2 homolog, is strikingly similar to the pore-forming domains of bacterial toxins. Though several Bcl-2 family members form pores in synthetic bilayers, we do not know whether they form pores in the native membrane and if they do, how their pore-forming activities contribute to the regulation of membrane permeability and apoptosis. Our long-term goal is to understand the molecular mechanisms by which Bcl-2 regulates apoptosis. Our short-term goal is to determine the functional, native Bcl-2 structure at and in the organelle membrane. We generated a series of fluorescent, full-length Bcl-2 proteins that each had a single dye attached at a specific site. The dye-labeled Bcl-2 proteins form pores and change spectra upon insertion into membranes. Hence the Bcl-2-membrane interaction can be detected and analyzed spectroscopically. Using the active, site-specifically labeled fluorescent Bcl-2 and a combination of fluorescence spectroscopic methods, we aim to characterize the structure of Bcl-2 at the ER microsomes and the mitochondria with focus on its transmembrane domain, pore and cytosolic domain. The results of the project will establish a structural basis for understanding the functions of Bcl-2 in regulating both membrane permeability and apoptosis, which is a prerequisite for designing therapeutic approaches to control Bcl-2 activity, to treat and perhaps to cure apoptotic diseases.